Numerous techniques for the detection of antibodies exist. These are utilized in many applications to determine the presence of any, or given, antibodies and to measure their concentration in a variety of fluids, most particularly blood. These techniques are particularly useful for typing erythrocytes and in the cross-matching of blood for transfusion.
The main technique commonly used in blood banking is called the antiglobulin test (AGT) which is performed by incubating a serum or plasma which is being tested for the presence of an antibody, with a suspension of the red blood cells for 30 minutes at 37° C. This phase is required to allow the binding of the antibodies, if present in the test serum, to the red cells. In the second phase, the sensitized red cells are washed three times with an isotonic solution of sodium chloride to remove the free serum proteins including free immunoglobulins. In the final phase, the washed red cells are mixed with a solution of antibody against human immunoglobulins, centrifuged and the red cell pellets are examined for aggregation, which would result from the reaction of the antiglobulin reagent with the red cell-bound antibodies, if present. For red cell typing, the same procedure is followed except that the test red cells are incubated with a known antibody used for typing. The completion of the AGT requires a total of 35 to 45 minutes, a time that is unacceptable for emergency situations
Another test commonly used is The Manual Polybrene Test (MPT) which is rapid, simple and economical. The processes involved can be divided into three phases, Namely the Sensitization Phase in which the antigen-antibody reaction takes place in a low ionic medium (LIM); the Polybrene-induced aggregation phase in which the sensitized erythrocytes are non-specifically aggregated by Polybrene, a positively charged synthetic polymer, and the Polybrene-Neutralization Phase in which a salt such as trisodium citrate is used to disperse the aggregates produced by polybrene. The occurrence of an antigen-antibody reaction is recognized by persistence of agglutination after the Polybrene effects have been reversed. Although the MPT is more sensitive than other existing tests for detection of many erythrocyte antibodies, it has been found to lack adequate sensitivity for Kell-related antibodies, a deficiency that has prevented MPT from becoming a universal blood bank test in the countries and populations where Kell-incompatibility is relatively common. This invention describes a new procedure which, unlike the AGT, is rapid, does not require red cell washing after red cell sensitization, and unlike the MPT, is highly sensitive for detection of the Kell related and other antibodies. The most commonly employed prior art procedure based on the use of antiglobulin reagent is described in U.S. Pat. No. 4,436,825. The antiglobulin procedure is described in an article by Coombs et al at Vol. 26, page 255 of the Brit. J. Exp. Path (1945). These procedures, however, have the drawbacks of being time-consuming and of inadequate sensitivity.
Another method is an automated method described in an article in Transfusion, Vol. 8, No. 6 November-December 1968 by P. Lalezari. That method utilizes another approach, however, that method has substantial drawbacks. It involves careful attention to proportions of ingredients and reagents as well as complicated equipment. Further, it too is time-consuming.
The disclosures of these articles are incorporated herein by reference. The drawbacks of these existing techniques pose substantial impediments to the need for fast and simple antibody detection with acceptable accuracy. The need for improved antibody detection techniques is therefore clear.
The new test is based on the discovery that macromolecules which are water soluble polymers when used in an amount that potentiates the agglutination of erythrocytes in the presence of antibodies or erythrocytes coated with antibodies. The macromolecules include sodium salts of various polycarboxylic acid macromolecules (hereafter PCA) such as poly[(isobutylene-alt-maleic acid)NH4-co(isobutylene-alt-maleic anhydride)] (hereafter “PIMA)”; various polyacrylic acids (PAA), carboxymethyl celluloses (CMC), polyvinyl pyrrolidone (PVP), and polymers of negatively charged amino acids (polyglutamic acid) of appropriate molecular sizes will react with immunoglobulins and will cause the immunoglobulins to precipitate. It has also been found that 2-hydroxyethyl cellulose with a weight average molecular weight of 90,000 Dalton is effective as a macromolecule at a concentration of this molecule was 0.75 w/v % in 2.5 w/v % dextrose. Another example of a macromolecule is hydroxypropyl cellulose with a weight average MW of 80,000 Dalton. The effective concentration of this molecule was between 2 to 3 w/v % in 2.5 w/v % dextrose. At these concentrations, these molecules potentiated the reaction of “complete” anti-M and anti-Kell antibodies. Other useful neutral macromolecules include Ficoll 400, Dextran T70, methyl cellulose (producing about 4,000 cP viscosity at 2% solution in water) and the like. These macromolecules when used at low concentrations with dilute systems did not cause red cell aggregation but caused the antibody-dependent red cell agglutinations produced by undiluted antibodies to become stronger and more stable. Examples of this type of effects are seen with polyethylene glycols (PEG) with weight average molecular weights of 8,000 and 20,000 Daltons. When these PEGs are used at concentrations between 5 to 10 w/v %, they stabilize the reaction of undiluted anti-M or anti Kell antibodies as used in Example I but they do not potentiate the reaction with diluted antibodies.
Neutral macromolecules such as 2-Hydroxyethyl cellulose and hydroxypropyl cellulose may be used at concentrations of 5 to 10% w/v slightly potentiate the reaction of warm antibodies as described herein but they are much less effective as compared to PCA or PVP.
If erythrocytes are coated with anti-erythrocyte antibodies, the addition of an effective amount of these molecules will cause the erythrocytes also to aggregate. It has also been discovered that polyvinylpyrrolidones (PVP) of appropriate molecular sizes also react with red cell-bound antibodies and cause their aggregation. The reactivity of large PVP molecules is suggested to be due to their weak positive charge which binds to the negatively charged moieties on immunoglobulin molecules. In this invention, the need for washing the sensitized red cells, as required in the standard antiglobulin test, is eliminated because the amount of effective macromolecules used are in excess of what may be neutralized by the proteins in the test plasmas. The PCA and PVP induced aggregation of erythrocytes which have antibodies bound to the surface, may be used as a test for the presence of anti-erythrocyte antibodies in a sample of serum or plasma, or for red cell typing. These tests are widely used in blood banks for red cell typing, for detection of anti-erythrocyte antibodies in collected whole blood, for cross-matching prior to the release of whole blood for transfusion or for the preparation of blood derived products.
By virtue of their ability to create a viscous environment, macromolecules are known to cause a reversible and nonspecific red cell aggregation known as rouleaux formation. In previous art, PVP was used as a rouleaux forming agent, to replace centrifugation, in a continuous flow system (Sturgeon et al. Vox Sang. 8:438, 1963). The ability to act as an antiglobulin agent described in this invention, however, is the newly recognized property of the PVP molecule. Neutral macromolecules such as polyethyleneglycols cause rouleaux formation but unlike PCA or PVP do not have antiglobulin activity.